For vitrification solutions to be biologically applicable, the cryoprotectants that comprise the vitrification solution must be contained within a “carrier” or “vehicle” solution used to provide osmotic and physiological support for living systems in the presence and absence of the cryoprotectants. However, it is well known in the art that the efficacy of carrier solutions for cryoprotectants is unpredictable and that the best carrier solution for one cryoprotectant or cryoprotectant mixture may be different from the best carrier solution for another cryoprotectant or cryoprotectant mixture.
As disclosed in Fahy, et al., U.S. patent application Ser. No. 09/400,793, filed Sep. 21, 1999, glucose inhibits the action of the polyvinyl alcohol type “ice blocking” (antinucleating) agent. This renders Euro-Collins solution (containing 190 mM glucose) or RPS-2 (containing 180 mM glucose) sub-optimal for use with such antinucleating agents. However, the inclusion of such agents, typified most fully by a product called “X1000, ” which is commercially available from 21st Century Medicine, Rancho Cucamonga, Calif. 91730, is highly desirable. Several alternative carrier solutions were disclosed in Fahy, et al. U.S. patent application Ser. No. 09/400,793, filed Sep. 21, 1999, issued as U.S. Pat. No. 6,395,467 B1 (herein incorporated by reference), such as MHP-2, GHP-2, and RPS-T. However, none of these was fully satisfactory. These other carriers provided poorer recovery of tissues maintained in them in the presence of vitrifiable concentrations of cryoprotectant than does RPS-2, or are both prohibitively expensive (RPS-T) and may be less biologically acceptable than RPS-2.
The difficulties of not having an excellent carrier solution are multiplied when the object is to vitrify massive structures such as natural organs or tissue engineered products such as artificial organs or tissues. The only relevant experience known in the art has been the use of either RPS-2 (Fahy and Ali, Cryobiology, 35:114-131, 1997) or Euro-Collins solution (Khirabadi and Fahy, Transplantation 70:51-57, 2000; Khirabadi and Fahy, Cryobiology 31:10-25, 1994; Arnaud, Fahy, and Khirabadi, Cryobiology 35: 358, 1997, and paper submitted for publication 2001) for the perfusion of rabbit kidneys with a vitrification solution called VS4 (formula defined in those citations). Without the ability to use either Euro-Collins or RPS-2 as a carrier solution, the practitioner is unable to rely on the state of the art in selecting a carrier solution for use, particularly given the extreme desirability of using a vitrification solution other than VS4 or its more concentrated relative, VS41A (formula published in, for example, G. M. Fahy et al., Chapter 20, in “Cell Biology of Trauma” (J. J. Lemasters and C. Oliver, Eds.), CRC Press, Boca Raton, Fla., 1995, pp. 333-356). This lack of a suitable carrier solution is a major impediment to applying vitrification to whole organs and engineered systems. This is particularly true when one considers the potential differential response of the organ parenchyma and the organ vasculature to a particular untested combination of cryoprotectants and carrier solution.